Correlation of SARS-CoV-2 Nucleocapsid Antigen and RNA Concentrations in Nasopharyngeal Samples from Children and Adults Using an Ultrasensitive and Quantitative Antigen Assay

Abstract

Diagnosis of COVID-19 by PCR offers high sensitivity, but the utility of detecting samples with high cycle threshold (C_T ) values remains controversial. Currently available rapid diagnostic tests (RDTs) for SARS-CoV-2 nucleocapsid antigens (Ag) have sensitivity well below PCR. The correlation of Ag and RNA quantities in clinical nasopharyngeal (NP) samples is unknown. An ultrasensitive, quantitative electrochemiluminescence immunoassay for SARS-CoV-2 nucleocapsid (the MSD S-PLEX SARS-CoV-2 N assay) was used to measure Ag in clinical NP samples from adults and children previously tested by PCR. The S-PLEX Ag assay had a limit of detection (LOD) of 0.16 pg/ml and a cutoff of 0.32 pg/ml. Ag concentrations measured in clinical NP samples (collected in 3.0 ml of media) ranged from less than 160 fg/ml to 2.7 μg/ml. Log-transformed Ag concentrations correlated tightly with C_T values. In 35 adult and 101 pediatric PCR-positive samples, the sensitivities were 91% (95% confidence interval, 77 to 98%) and 79% (70 to 87%), respectively. In samples with a C_T of ≤35, the sensitivities were 100% (88 to 100%) and 96% (88 to 99%), respectively. In 50 adult and 40 pediatric PCR-negative specimens, the specificities were 100% (93 to 100%) and 98% (87 to 100%), respectively. Nucleocapsid concentrations in clinical NP samples span 8 orders of magnitude and correlate closely with RNA concentrations (C_T values). The S-PLEX Ag assay showed 96 to 100% sensitivity in samples from children and adults with C_T values of ≤35, and a specificity of 98 to 100%. These results clarify Ag concentration distributions in clinical samples, providing insight into the performance of Ag RDTs and offering a new approach to diagnosis of COVID-19.

Keywords: COVID-19; SARS-CoV-2; antigen; diagnostic; nucleoprotein.

FIG 1

Nucleocapsid concentrations in clinical NP swab samples from PCR-negative (green) and PCR-positive (blue) adults (filled circles) and pediatric patients (Ped, open circles). The figure shows the concentrations measured using the optimal assay format and dilution for each sample as described in Materials and Methods. Concentrations below the LOD for the S-PLEX assay were assigned the LOD value (gray dashed line). The plots also show dashed lines to indicate the applied assay thresholds for the ultrasensitive S-PLEX and conventional R-PLEX ECL assays, as well as the estimated analogous values for the commercial BD Veritor and Quidel Sofia systems.

FIG 2

Correlation of nucleocapsid concentration with PCR C_T value for clinical NP swab samples from PCR-positive adults (red circles) and pediatric patients (Ped, blue circles). The figure shows the concentrations measured using the optimal assay format and dilution for each sample as described in Materials and Methods. Concentrations below the LOD for the S-PLEX assay were assigned the LOD value (gray dashed horizontal line). The diagonal dashed gray line is the linear regression fit to the data (using log-transformed nucleocapsid concentrations). To provide estimates of the expected C_T value for samples at the threshold for different nucleocapsid assay formats, the plots also show horizontal dashed lines to indicate the applied assay thresholds for the ultrasensitive S-PLEX and conventional R-PLEX ECL assays, as well as the estimated analogous values for the commercial BD Veritor and Quidel Sofia systems.

FIG 3

(a and b) Correlation of nucleocapsid concentration (log₂ tranformed) (a) and PCR C_T value (b) with time from symptom onset for clinical NP swab samples from PCR-positive adults (red circles) and pediatric patients (Ped, blue circles). The plots also show the linear regression fit to the data (diagonal dashed gray lines).